Electron transport pathways to nitrous oxide in Rhodobacter species

David J. Richardson, Alastair G. McEwan, J. Baz Jackson, Stuart J. Ferguson

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1. Electron transport components involved in nitrous oxide reduction in several strains of Rhodobacter capsulatus and in the denitrifying strain of Rhodobacter sphaeroides (f. sp. denitrificans) have been investigated. Detailed titrations with antimycin A and myxothiazol, inhibitors of the cytochrome bc1 complex, show that part of the electron flow to nitrous oxide passes through this complex. The sensitivity to myxothiazol varies between strains and growth conditions of R. capsulatus; the higher rates of nitrous oxide reduction correlate with the higher sensitivities. Partial inhibition of the nitrous oxide reductase enzyme with azide decreased the sensitivity to myxothiazol of the strains that had the highest nitrous oxide reductase activity. 

2. Inhibition of nitrous oxide reduction in cells of R. capsulatus by myxothiazol could be restored under dark conditions by addition of N,N,N′,N′‐tetramethyl‐p‐phenylene diamine. The highest activities observed after addition of this electron carrier were found in the strains that had the highest sensitivity to myxothiazol, consistent with the premise that this inhibitor is more effective at the higher flux rates to nitrous oxide. 

3. Addition of nitrous oxide to cells of R. capsulatus strain N22DNAR+ under darkness caused oxidation of both b‐ and c‐type cytochromes. The oxidation of b cytochromes was less pronounced in the presence of myxothiazol, consistent with a role for the cytochrome bc1 complex in the electron pathway to nitrous oxide. Ferricyanide, in the absence of myxothiazol, caused a similar extent of oxidation of b cytochromes, but a greater oxidation of c‐type, suggesting that there was a pool of c‐type cytochrome that was not oxidisable by nitrous oxide. The time course showed that both the b‐ and c‐type cytochromes were oxidised within a few seconds of the addition of nitrous oxide. During the following seconds there was a partial re‐reduction of the cytochromes such that after approximately 1 min a lower steady‐state of oxidation was attained and this persisted until the nitrous oxide was exhausted. 

4. A mutant, MTCBC1, of R. capsulatus that specifically lacked a functional cytochrome bc1 complex reduced nitrous oxide, albeit at 30% of the rate shown by the parent strain MT1131. A reduced minus nitrous‐oxide‐oxidised difference spectrum for MTCBC1 in the absence of myxothiazol was similar to the corresponding difference spectrum observed for strain N22DNAR+ in the presence of myxothiazol. It is suggested that these difference spectra identify the cytochrome components, including a b‐type, involved in a pathway that is alternative to, and independent of, the cytochrome bc1 complex. It is concluded that antimycin‐A‐ or myxothiazol‐insensitive nitrous oxide reduction in strains of R. capsulatus that possess the cytochrome bc1 complex can originate from: (a) the operation of the alternative pathway identified in mutant MTCBC1; (b) a leak of electrons past the binding sites for these inhibitors in the cytochrome bc1 complex; (c) a combination of these two contributions. 

5. 2‐n‐Heptyl‐4‐hydroxyquinoline N‐oxide inhibited the pathway of electron transport to nitrous oxide in R. capsulatus that was independent of the cytochrome bc1 complex. Oxidation by nitrous oxide of b‐ and c‐type cytochromes involved in this pathway was not affected by the inhibitor. It is suggested that 2‐n‐heptyl‐4‐hydroxyquinoline N‐oxide blocks the ubiquinol oxidation step in the cytochrome‐bc1‐independent pathway. 

6. Although several lines of evidence indicate that the cytochrome bc1 complex can participate in electron flow to nitrous oxide, a variety of data confirm that electron flow to other anaerobic electron acceptors, nitrate, trimethylamine N‐oxide and dimethylsulphoxide, in R. capsulatus does not involve this complex.

Original languageEnglish
Pages (from-to)659-669
Number of pages11
JournalEuropean Journal of Biochemistry
Issue number3
Publication statusPublished - Nov 1989

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